GSM6809973: JM8.N4 mESC, Triptolide 45min, RCMC, rep2; Mus musculus; OTHER
Cells were fixed with 3 mM DSG for 35 minutes, followed by an additional 10 minutes with 1% formaldehyde. Following quenching, nuclei were prepared from fixed cells. Chromatin was digested in situ using MNase, DNA 3' ends were chewed back and biotinylated, and then free DNA ends were proximity ligated. Unligated ends were digested, and followed by reverse crosslinking of the chromatin and Protease K digestion. DNA was purified by phenol chloroform extraction and DNA Clean and Concentrator kit (Zymo), and then size selected for dinucleosomal fragments on an agarose gel. Following end polishing of DNA fragments, a streptavidin pulldown was used to enrich for ligated dinucleosomal fragments. Size selected DNA fragments bound to streptavidin coated beads were used to prepare Micro-C libraries using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB), using NEBNext Multiplex Oligos for Illumina for indexing samples. Libraries were amplified with 7-8 PCR cycles on the thermocycler using KAPA HiFi HotStart ReadyMix (Roche) and purified using AMPure XP beads (Beckman Coulter). Purified libraries were quantified using qPCR and pooled in a 1:1 ratio to a total of 2-4 µg of DNA. Following the Twist Standard Hybridization Target Enrichment Protocol (Twist Biosciences) with the inclusion of Mouse Cot-1 DNA (Invitrogen) blocker solution, this library pool was hybridized to custom probe panels for loci of interest and bound to streptavidin coated beads. Bead-bound fragments were pulled down, amplified with 6 PCR cycles on the thermocycler using Equinox Library Amp Mix (Twist), and purified using Twist Binding and Purification Beads (Twist). Following qPCR quantification, the target enriched library pool was submitted for sequencing.